Project Summary/Abstract Mucormycosis, caused by Mucorales fungi, is a life-threatening infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), neutropenia, corticosteroid use, and/or increased serum iron. Rhizopus oryzae is the most common cause of mucormycosis. Because of the rising prevalence of mucormycosis risk factors, the incidence of the infection has risen. Despite disfiguring surgery and aggressive antifungal therapy, the mortality of mucormycosis remains >40%, and approaches 100% in patients with disseminated disease. Clearly new strategies to prevent and treat mucormycosis are needed. Mucormycosis is commonly initiated via inhalation. Primary infection sites of rhino-orbital disease is almost exclusively reported in DKA patients, while those with hematologic malignancies suffer from pulmonary mucormycosis. Regardless of the initial infected site, a clinical hallmark of mucormycosis is the virtually uniform presence of extensive angioinvasion with resultant vessel thrombosis and tissue necrosis. These features highlight the importance of the fungus ability to invade nasal or alveolar epithelial cells to initiate the infection. They also emphasize the significance of the endothelium penetration during progression of the disease into hematogenously disseminated mucormycosis. During the last funding cycle, we elucidated that R. oryzae invades endothelial cells via a unique interaction between the fungal CotH3 (this protein is present only in Mucorales) and the host receptor GRP78. Both cell surface proteins are upregulated in physiological conditions found in DKA patients such as hyperglycemia, elevated iron and ketone bodies. We recently identified GRP78 as a putative receptor on nasal epithelial cells also, while the alveolar epithelial cell receptor(s) is likely to be neuropilins and/or integrins. These findings add to our understanding why rhino-orbital disease occurs in DKA patients rather than pulmonary infection. Equally important, we successfully purified two unique protein toxins (ricin-like and Type X secretion system) that cause damage to host cells in vitro and death to mice upon injection. These two toxins are highly likely responsible for the clinical hallmark of vessel thrombosis and tissue necrosis associated with antifungal therapy failure. Thus, we propose to build on these exciting data and: 1) define the molecular mechanisms by which R. oryzae binds to and invades nasal and alveolar epithelium during initiation of infection; and 2) characterize the role of toxins in the host cell invasion and mucormycosis pathogenesis as well as determine their mechanism of action. Our studies will: 1) further elucidate the role of host receptors and fungal ligands in initiation of mucormycosis; 2) determine how the two unique fungal toxins, discovered and partially characterized, in the previous funding cycle promote invasion and damage to host cells; and 3) facilitate the development of novel therapies targeting fungal ligands, host receptors, and toxins.